WO2024071366A1 - Root nodule formation-promoting composition, and root hair formation-promoting composition - Google Patents

Abstract

The purpose of the present disclosure is to provide a root nodule formation-promoting composition capable of promoting the formation of root nodules.　One embodiment is a root nodule formation-promoting composition which contains: one or more biosurfactants selected from peptide biosurfactants and sugar biosurfactants; and an agriculturally acceptable carrier.

Description

Composition for promoting nodule formation and composition for promoting root hair formation

The present disclosure relates to a composition for promoting nodule formation and a composition for promoting root hair formation.

It is known that the yield of legume plants increases when they form many nodules. For example, Patent Document 1 discloses a method for increasing nodules by applying a composition containing a compound (A) such as stearyl alcohol to a plant that forms nodules. Patent Document 1 also discloses that the composition may contain a surfactant (B) for the purpose of emulsifying, dispersing, solubilizing, or promoting the penetration of compound (A).

Patent document 2 discloses a soil treatment composition for enhancing immune health, growth and/or yield of plants, the composition including Wickerhamomyces anomalus yeast and/or its growth by-products. The soil treatment composition of Patent document 2 is disclosed to be a microbial-based soil treatment composition for enhancing the health, growth and total yield of crop plants by enhancing the health and/or growth of the plant's root system and by stimulating the plant's innate immunity and other metabolic systems that contribute to plant health and productivity. Wickerhamomyces anomalus yeast is a yeast-like fungus that produces ethyl acetate, and is known to cause a paint thinner odor when attached to food, etc.

Patent Document 3 discloses a composition for reducing greenhouse gases, improving carbon utilization, and/or enhancing carbon sequestration, comprising one or more beneficial microorganisms and/or one or more microbial growth by-products, wherein the one or more beneficial microorganisms are selected from non-pathogenic yeasts, fungi, and bacteria, and the one or more growth by-products are selected from biosurfactants and enzymes. Patent Document 3 discloses that applying the composition to soil improves the soil environment and promotes plant growth.

Patent Document 4 discloses a legume plant growth promoter that contains soyasaponin and a nonionic surfactant with an HLB of 9 to 20 as active ingredients. It is disclosed that the legume plant growth promoter of Patent Document 4 enables an increase in the weight of the underground parts of legume plants (an increase in the dry weight of the underground parts).

JP 2008-162912 A JP 2022-509204 A JP 2022-504068 A JP 2020-033331 A

　The use of compositions to increase the number of nodules has been studied in the past, but for example, in Patent Document 1, only spraying of the composition on stems and leaves at the two-leaf stage or later was considered, and research has yet to be fully conducted.

The present disclosure therefore aims to provide a composition for promoting nodule formation that is capable of promoting the formation of nodules.

Furthermore, the promotion of root growth using compositions has been studied in the past. However, Patent Documents 1 to 4 do not study, describe or suggest anything about promoting the formation of root hairs.

The function of root hairs is to collect water and nutrients contained in the soil and distribute them through the roots to all parts of the plant, making them extremely important organs for plants. In legume plants, root hairs are also important organs for forming root nodules. However, while promotion of root growth has been studied in the past, promotion of root hair formation has not been studied at all. Furthermore, according to the inventors' studies, no clear correlation was found between promotion of root growth and promotion of root hair formation.

Therefore, another aspect of the present disclosure aims to provide a composition for promoting root hair formation that is capable of promoting the formation of root hairs.

An example of this embodiment is described as follows:

[1] A composition for promoting nodule formation, comprising at least one biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant, and an agriculturally acceptable carrier.
[2] The composition for promoting nodule formation according to [1], wherein the biosurfactant is contained in an amount of 0.001 to 2% by mass relative to 100% by mass of the composition for promoting nodule formation.
[3] The composition for promoting nodule formation according to [1] or [2], which contains a soybean meal hydrolyzate.
[4] The composition for promoting nodule formation according to [3], wherein the mass ratio of the biosurfactant to the soybean meal hydrolyzate (biosurfactant:soybean meal hydrolyzate) is 1:0.05 to 1:150.
[5] The composition for promoting nodule formation according to any one of [1] to [4], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[6] The composition for promoting nodule formation according to any one of [1] to [5], which is used for coating plant seeds with the biosurfactant or for irrigating soil in which a plant is grown.
[7] A precursor of a composition for promoting nodule formation, which becomes the composition for promoting nodule formation according to any one of [1] to [6] upon dilution.
[8] A seed treated with the composition for promoting nodule formation according to any one of [1] to [6].
[9] A composition for promoting root hair formation, comprising at least one biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant, and an agriculturally acceptable carrier.
[10] The composition for promoting root hair formation according to [9], wherein the biosurfactant is contained in an amount of 0.005 to 0.2% by mass based on 100% by mass of the composition for promoting root hair formation.
[11] The composition for promoting root hair formation according to [9] or [10], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[12] The composition for promoting root hair formation according to any one of [9] to [11], which is intended to be sprayed on plant leaves.
[13] A precursor of a composition for promoting root hair formation, which becomes the composition for promoting root hair formation according to any one of [9] to [12] upon dilution.
This specification includes the disclosures of Japanese Patent Application Nos. 2022-158013, 2022-158059, and 2023-056902, which are priority documents of this application.

The composition for promoting nodule formation of the present disclosure can promote the formation of root nodules. In addition, the composition for promoting root hair formation of the present disclosure can promote the formation of root hairs.

The experimental observations shown in Table 5 are shown in FIG. The experimental observations shown in Table 6 are shown in FIG. The experimental observations shown in Table 7 are shown in FIG. The experimental observations shown in Table 8 are shown in FIG. The experimental observations shown in Table 9 are shown in FIG. The experimental observations shown in Table 10 are shown in FIG.

The present invention will be described in detail below.
The composition for promoting root nodule formation according to the present embodiment includes at least one biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants, and an agriculturally acceptable carrier. The composition for promoting root hair formation according to the present embodiment includes at least one biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants, and an agriculturally acceptable carrier.

<Biosurfactants>
The composition for promoting nodule formation and the composition for promoting root hair formation contain a biosurfactant. Examples of the biosurfactant include at least one type of biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant. The composition for promoting nodule formation and the composition for promoting root hair formation may contain one type of biosurfactant alone or two or more types of biosurfactants. Since the composition for promoting nodule formation contains a biosurfactant, it can promote the formation of nodules by applying it to a plant.

The root hair formation promoting composition contains a biosurfactant, and thus can promote the formation of root hairs when applied to plants. The reason for this is unclear, but this effect was not observed when a synthetic surfactant was used, and is an effect unique to biosurfactants. Furthermore, the root hair formation promoting composition can exert its effect even if it is not applied directly to the roots, for example, by applying it to the leaves. For this reason, the root hair formation promoting composition can be easily applied to plants, and is therefore preferable.

In addition, in the composition for promoting nodule formation and the composition for promoting root hair formation, it is preferable to use a biosurfactant that is not derived from Wickerhamomyces anomalus yeast from the viewpoints of odor and food hygiene.

An example of a peptide-type biosurfactant is a lipopeptide biosurfactant. A lipopeptide biosurfactant has a peptide containing a hydrophobic group and a hydrophilic portion, exhibits surface activity, and is produced by a microorganism. Examples of lipopeptide biosurfactants include surfactin, arthrofactin, iturin, fengicin, serawettin, lykesin, viscosin, and salts thereof.

The peptide-type biosurfactant is preferably at least one peptide-type biosurfactant selected from surfactin and its salts. Surfactin and surfactin salts can be represented by the following general formula (1). As the surfactin and surfactin salts, one type or two or more types may be used.

［式（１）中、Ｘは、ロイシン、イソロイシンおよびバリンから選択されるアミノ酸の残基を示し、Ｒは炭素数９～１８のアルキル基を示し、Ｍ^＋はそれぞれ独立に、水素イオン（Ｈ⁺）、アルカリ金属イオン、アンモニウムイオン又はピリジニウムイオンを示す。］

[In formula (1), X represents an amino acid residue selected from leucine, isoleucine, and valine, R represents an alkyl group having 9 to 18 carbon atoms, and each M ⁺ independently represents a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion.]

In addition, when M ⁺ is a hydrogen ion, it means that _CO2- ⁽ M ⁺ ) is a hydroxyl group (COOH group). When two M ⁺ are hydrogen ions, it means surfactin, and when at least one M ⁺ is an alkali metal ion, ammonium ion or pyridinium ion, it means a salt of surfactin. The general formula of surfactin is shown below as general formula (1').

［式（１’）中、Ｘ及びＲは、式（１）と同義である］

[In formula (1′), X and R are defined as in formula (1)]

X is an amino acid residue selected from leucine, isoleucine and valine, but may be an L-amino acid residue or a D-amino acid residue, with an L-amino acid residue being preferred.

R is an alkyl group having 9 to 18 carbon atoms, and is a linear or branched monovalent saturated hydrocarbon group having 9 to 18 carbon atoms. Examples of alkyl groups having 9 to 18 carbon atoms include n-nonyl, 6-methyloctyl, 7-methyloctyl, n-decyl, 8-methylnonyl, n-undecyl, 9-methyldecyl, n-dodecyl, 10-methylundecyl, n-tridecyl, 11-methyldodecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, and n-octadecyl, with 10-methylundecyl being preferred.

Each M ⁺ is independently a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion. The alkali metal ion is not particularly limited, but may be a lithium ion, a sodium ion, a potassium ion, or the like. The ammonium ion is not particularly limited, but may be, for example, an ammonium ion represented by N(R ¹ ) ₄ ⁺ . Each R ¹ independently represents hydrogen or an organic group. As the ammonium ion, a quaternary ammonium ion in which all R ¹ are organic groups is one of the preferred embodiments. Examples of the organic group include an alkyl group, an aralkyl group, and an aryl group. Specifically, examples of the alkyl group include alkyl groups having 1 to 10 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, and t-butyl, examples of the aralkyl group include aralkyl groups having 7 to 12 carbon atoms such as benzyl, methylbenzyl, and phenylethyl, and examples of the aryl group include aryl groups having 6 to 15 carbon atoms such as phenyl, toluyl, and xylyl. Examples of the ammonium ion include, for example, a tetramethylammonium ion and a tetraethylammonium ion. Examples of the pyridinium ion are not particularly limited. In the pyridinium ion, the hydrogen atom bonded to the carbon atom constituting the pyridine ring may be substituted with an organic group. In addition, in the pyridinium ion, the N ⁺ constituting the pyridine ring may be bonded to, for example, hydrogen or an organic group. In addition, as the organic group possessed by the pyridinium ion, the organic groups listed in the description of ^R1 can be appropriately used.

The two M ⁺ present in the general formula (1) may be the same or different from each other. As for the two M ⁺ present in the general formula (1), for example, a part of M ⁺ is a hydrogen ion, and a part of M ⁺ is an alkali metal ion, which is one of the preferred embodiments. The alkali metal ion is not particularly limited, but represents a lithium ion, a sodium ion, a potassium ion, or the like. In addition, when the two M ⁺ present in the general formula (1) are two or more types of ions, when focusing on a certain molecule (salt), the two M ⁺ may be the same type of ion. When M ⁺ is two types of ions, the ratio (molar ratio) of a certain ion A to a certain ion B is, for example, 1:10 to 10:1, preferably 1:5 to 5:1, and more preferably 1:3 to 3:1. It is one of the preferred embodiments that a part of the two M ⁺ present in the general formula (1) is a hydrogen ion, and a part is a sodium ion (Na ⁺ ).

Peptide-type biosurfactants such as surfactin or a salt of surfactin can be obtained by culturing a microorganism, for example a strain belonging to Bacillus subtilis, and isolating it from the culture medium according to known methods. A purified product may be used, or it may be used unpurified, for example in the culture medium. Furthermore, those obtained by chemical synthesis methods may be used in the same way as long as the molecular structure is the same. Commercially available products may also be used.

Glycosurfactants include rhamnolipids, sophorolipids, mannosylerythritol lipids, cellobiose lipids, trehalose lipids, succinoyl trehalose lipids, glucose lipids, polyol lipids, oligosaccharide fatty acid esters, and salts thereof.

The glycosurfactant is preferably at least one glycosurfactant selected from rhamnolipids, sophorolipids, and salts thereof.

Glycosurfactants can be obtained according to known methods. Commercially available products can also be used.

The biosurfactant is preferably at least one selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.

<Soybean meal hydrolyzate>
The composition for promoting nodule formation may contain a soybean meal hydrolyzate. When the composition for promoting nodule formation contains a soybean meal hydrolyzate, it is preferable because the composition can promote nodule formation even when the concentration of the biosurfactant is low.

Soybean meal hydrolyzate can be obtained, for example, by subjecting soybean meal to a Bacillus fermentation treatment using Bacillus bacteria.

Agriculturally acceptable carriers
The composition for promoting root nodule formation and the composition for promoting root hair formation include an agriculturally acceptable carrier. In the present disclosure, the agriculturally acceptable carrier may be any carrier capable of holding the biosurfactant, and may be a liquid carrier or a solid carrier. The solid carrier may be a hydratable solid material. The solid carrier may be in the form of a powder or granules.

The agriculturally acceptable carrier is preferably a liquid carrier such as water or an organic solvent. Here, the water as a carrier is not limited to pure water, but may be an aqueous solution, an aqueous suspension, an aqueous gel, or an aqueous slurry, and may have viscosity. Similarly, the organic solvent is not limited to a pure organic solvent, but may be an organic solvent-based solution, a suspension, a gel, or a slurry, and may have viscosity. Examples of organic solvents include methyl ether, ethyl ether, propyl ether, and butyl ether.

The agriculturally acceptable carrier is preferably a liquid carrier such as water or an aqueous solution in which a hydratable substance is dissolved in water, or a solid carrier containing a hydratable substance that can be dissolved in water. Examples of hydratable substances include polyvinylpyrrolidone, random and block copolymers of alkylene oxides, vinyl acetate/vinylpyrrolidone copolymers, alkylated vinylpyrrolidone copolymers, polyalkylene glycols including polypropylene glycol and polyethylene glycol, polyvinyl acetate, polyvinyl alcohol, gelatin, agar, gum arabic, karaya gum, tragacanth gum, guar gum, locust bean gum, xanthan gum, ghatti gum, carrageenan, alginates, casein, dextran, pectin, chitin, 2-hydroxyethyl starch, 2-aminoethyl starch, 2-hydroxyethyl cellulose, methylcellulose, carboxymethyl cellulose salts, cellulose sulfate, polyacrylamide, alkali metal salts of maleic anhydride copolymers, and alkali metal salts of poly(meth)acrylates.

<Additives>
The composition for promoting nodule formation may contain, as necessary, components other than the biosurfactant, the soybean meal hydrolyzate, and the agriculturally acceptable carrier as additives. The composition for promoting nodule formation may contain one type of additive, or two or more types of additives. Examples of additives include, but are not limited to, moisturizers, colorants, antifoaming agents, UV protection agents, antifreeze agents, preservatives, biological control agents or biocides, emulsifiers, bulking agents, scavengers, plasticizers, phospholipids, flow agents, fusion aids, waxes, root nodule bacteria materials, and/or fillers (e.g., clay, talc, glass fiber, cellulose, finely powdered wood, etc.).

The composition for promoting root hair formation may contain, as necessary, components other than the biosurfactant and agriculturally acceptable carrier as additives. The composition for promoting root hair formation may contain one type of additive, or two or more types of additives. Additives include, but are not limited to, moisturizers, colorants, defoamers, UV protectants, antifreeze agents, preservatives, biological control agents or biocides, emulsifiers, bulking agents, scavengers, plasticizers, phospholipids, flow agents, coalescing agents, waxes, and/or fillers (e.g., clay, talc, glass fiber, cellulose, micronized wood, etc.).

<Composition for promoting nodule formation>
The composition for promoting nodule formation contains at least one type of biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants, as described above, and an agriculturally acceptable carrier.

In this disclosure, "substantially free" of a certain component means that the component is present in an amount of 0.01 parts by mass or less, preferably 0.001 parts by mass or less, when the biosurfactant contained in the composition for promoting nodule formation is taken as 100 parts by mass.

The composition for promoting nodule formation preferably contains 0.001 to 2% by mass of the biosurfactant, and more preferably 0.00125 to 1% by mass, based on 100% by mass of the composition for promoting nodule formation. In addition, when the composition for promoting nodule formation does not substantially contain soybean meal hydrolyzate, the composition for promoting nodule formation preferably contains 0.001 to 2% by mass of the biosurfactant, and more preferably 0.00125 to 1% by mass, based on 100% by mass of the composition for promoting nodule formation. This is preferable because the formation of nodules can be particularly effectively promoted within the above range.

In one preferred embodiment, the composition for promoting nodule formation contains the biosurfactant and a soybean meal hydrolysate. When the composition for promoting nodule formation contains the biosurfactant and a soybean meal hydrolysate, the mass ratio of the biosurfactant to the soybean meal hydrolysate (biosurfactant:soybean meal hydrolysate) is preferably 1:0.05 to 1:150, and more preferably 1:0.1 to 1:100. In addition, when the composition for promoting nodule formation contains the biosurfactant and a soybean meal hydrolyzate, it is preferable that the biosurfactant is contained in an amount of 0.005 to 0.2% by mass in 100% by mass of the composition for promoting nodule formation, and the soybean meal hydrolyzate is contained in an amount of 0.005 to 1% by mass in 100% by mass of the composition for promoting nodule formation, and it is more preferable that the biosurfactant is contained in an amount of 0.00625 to 0.1% by mass in 100% by mass of the composition for promoting nodule formation, and the soybean meal hydrolyzate is contained in an amount of 0.01 to 0.625% by mass in 100% by mass of the composition for promoting nodule formation. Within the above ranges, it is preferable because the formation of nodules can be particularly effectively promoted.

When the composition for promoting nodule formation is substantially free of soybean meal hydrolysate and is a composition for coating plant seeds with the biosurfactant, it preferably contains 0.05 to 75 g of the biosurfactant per ton of seeds, and more preferably 0.1 to 50 g. Also, when the composition for promoting nodule formation is substantially free of soybean meal hydrolysate and is a composition that is applied directly to roots by irrigating soil or mixing with water in hydroponic cultivation, it is preferable to contain 0.005 to 2% by mass of the biosurfactant in 100% by mass of the composition for promoting nodule formation, and more preferably 0.01 to 1% by mass. This range is preferable because it is particularly effective in promoting the formation of nodules.

When the composition for promoting nodule formation contains a biosurfactant and a soybean meal hydrolysate and is a composition for coating plant seeds with the biosurfactant and the soybean meal hydrolysate, it preferably contains 0.25 to 10 g of the biosurfactant per ton of seeds, and more preferably 0.5 to 5 g. Also, when the composition for promoting nodule formation contains a biosurfactant and a soybean meal hydrolysate and is a composition that is applied directly to roots by irrigating soil or mixing with water in hydroponic cultivation, it is preferable that the biosurfactant be contained in an amount of 0.005 to 0.2% by mass, and more preferably 0.01 to 0.1% by mass, based on 100% by mass of the composition for promoting nodule formation. This range is preferable because it is particularly effective in promoting the formation of nodules.

There are no particular limitations on the method for producing the composition for promoting nodule formation. For example, when the agriculturally acceptable carrier is a liquid carrier, a method in which a biosurfactant and optional additives, or a biosurfactant, a soybean meal hydrolyzate and optional additives are added to the liquid carrier and stirred until homogenous can be mentioned.

The composition for promoting nodule formation is usually applied to plants, soil, or water given to plants in hydroponic cultivation, etc., and is used, for example, on plant seeds, etc., or on soil. In one preferred embodiment, the composition for promoting nodule formation is a composition for coating plant seeds with the biosurfactant, or for irrigating soil in which plants are grown.

<Precursor of composition for promoting nodule formation>
An embodiment of the present disclosure includes a precursor of a composition for promoting nodule formation, which becomes the above-mentioned composition for promoting nodule formation upon dilution. The dilution is usually performed with the above-mentioned liquid carrier. The precursor of a composition for promoting nodule formation usually contains a biosurfactant and an agriculturally acceptable carrier, and the concentration of the biosurfactant is higher than that of the composition for promoting nodule formation. When a composition for promoting nodule formation is prepared using such a precursor of a composition for promoting nodule formation, it is preferable because a uniform composition for promoting nodule formation can be easily prepared.

The nodule formation promoting composition precursor preferably contains a biosurfactant at a concentration 10 to 100,000 times higher than that of the nodule formation promoting composition. When the nodule formation promoting composition contains a soybean meal hydrolyzate, the nodule formation promoting composition precursor preferably contains a soybean meal hydrolyzate at a concentration 10 to 100,000 times higher than that of the nodule formation promoting composition. The biosurfactant contained in the nodule formation promoting composition precursor is preferably 50% by mass or less, more preferably 20% by mass or less, based on 100% by mass of the nodule formation promoting composition precursor. The agriculturally acceptable carrier (preferably a liquid carrier) contained in the nodule formation promoting composition precursor is preferably 20% by mass or more, more preferably 40% by mass or more, based on 100% by mass of the nodule formation promoting composition precursor.

<Plant cultivation methods and seeds>
An embodiment of the present disclosure includes a method for cultivating a plant, in which the above-mentioned composition for promoting nodule formation is applied to the plant or soil. Also, an embodiment of the present disclosure includes a seed treated with the above-mentioned composition for promoting nodule formation. More specifically, a seed is preferred in which the surface of the seed is coated with a biosurfactant by contacting the seed with the composition for promoting nodule formation. It is also preferred to irrigate the soil in which the plant is cultivated with the composition for promoting nodule formation.

When the surface of the seeds is coated with a biosurfactant, it is preferable to use the composition for promoting nodule formation in an amount that results in a biosurfactant content of preferably 0.05 to 75 g, more preferably 0.1 to 50 g, per ton of seeds. In the present invention, ton (also written as t) means metric ton, i.e. 1 ton means 1000 kg. More specifically, when the composition for promoting nodule formation does not substantially contain soybean meal hydrolyzate, it is preferable to use the composition for promoting nodule formation in an amount that results in a biosurfactant content of preferably 0.05 to 75 g, more preferably 0.1 to 50 g, per ton of seeds. When the composition for promoting nodule formation contains a biosurfactant and soybean meal hydrolyzate, it is preferable to use the composition for promoting nodule formation in an amount that results in a biosurfactant content of preferably 0.25 to 10 g, more preferably 0.5 to 5 g, per ton of seeds. When the composition for promoting nodule formation contains the biosurfactant and soybean meal hydrolysate, the mass ratio of the biosurfactant to the soybean meal hydrolysate (biosurfactant:soybean meal hydrolysate) is preferably 1:5 to 1:125, and more preferably 1:10 to 1:100.

When the composition for promoting nodule formation is applied to soil, it is preferable to use the composition in an amount that provides a biosurfactant of preferably 0.0001 to 0.1 g, more preferably 0.00043 to 0.043 g per plant. More specifically, when the composition for promoting nodule formation does not substantially contain a soybean meal hydrolyzate, it is preferable to use the composition in an amount that provides a biosurfactant of preferably 0.0001 to 0.1 g, more preferably 0.00043 to 0.043 g per plant. When the composition for promoting nodule formation contains a biosurfactant and a soybean meal hydrolyzate, it is preferable to use the composition in an amount that provides a biosurfactant of preferably 0.0001 to 0.01 g, more preferably 0.00043 to 0.043 g per plant. When the composition for promoting nodule formation contains the biosurfactant and soybean meal hydrolysate, the mass ratio of the biosurfactant to the soybean meal hydrolysate (biosurfactant:soybean meal hydrolysate) is preferably 1:0.05 to 1:20, and more preferably 1:0.1 to 1:10.

When applying the composition for promoting nodule formation to soil, there are no particular limitations on the timing of application (irrigation), but it is preferable to apply it at the same time as plant sowing or after sowing and up until the three-leaf stage.

<Plants>
In the present disclosure, the plant is not particularly limited as long as it forms nodules, but is preferably a legume, such as soybean, peanut, pea, kidney bean, adzuki bean, broad bean, sweet pea, and astragalus.

<Composition for promoting root hair formation>
The composition for promoting root hair formation contains at least one biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants, as described above, and an agriculturally acceptable carrier.

The composition for promoting root hair formation preferably contains 0.005 to 0.2% by mass of the biosurfactant, and more preferably 0.01 to 0.1% by mass, based on 100% by mass of the composition for promoting root hair formation. When the composition for promoting root hair formation is a composition for spraying on plant leaves, the composition for promoting root hair formation preferably contains 0.01 to 0.2% by mass of the biosurfactant, and more preferably 0.02 to 0.1% by mass, based on 100% by mass of the composition for promoting root hair formation. When the composition for promoting root hair formation is a composition for direct application to roots by irrigating soil or mixing with water in hydroponic cultivation, the composition for promoting root hair formation preferably contains 0.0005 to 0.05% by mass of the biosurfactant, and more preferably 0.01 to 0.02% by mass, based on 100% by mass of the composition for promoting root hair formation. The above range is preferable because it is possible to particularly effectively promote root hair formation.

There are no particular limitations on the method for producing the composition for promoting root hair formation. For example, when the agriculturally acceptable carrier is a liquid carrier, a method in which a biosurfactant and any additives used are added to the liquid carrier and stirred until homogenous can be mentioned.

The root hair formation promoting composition is usually applied to plants, soil, or water given to plants in hydroponics, etc., and is used, for example, on plant leaves, seeds, seedlings, fruits, etc., or on soil. In one preferred embodiment, the root hair formation promoting composition is sprayed on the leaves of a plant. Spraying on the leaves of a plant means spraying at least the leaves, and may also include spraying on stems and leaves, and may further include spraying a portion on flowers, fruits, etc.

<Composition precursor for promoting root hair formation>
As an embodiment of the present disclosure, a root hair formation promoting composition precursor that becomes the above-mentioned root hair formation promoting composition by dilution can be mentioned. The dilution is usually performed with the above-mentioned liquid carrier. The root hair formation promoting composition precursor usually contains at least one biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant, and an agriculturally acceptable carrier, and the concentration of the biosurfactant is higher than that of the root hair formation promoting composition. When the root hair formation promoting composition is prepared using such a root hair formation promoting composition precursor, it is preferable because it is possible to easily prepare a uniform root hair formation promoting composition.

The root hair formation promoting composition precursor preferably contains a biosurfactant at a concentration 10 to 20,000 times higher than that of the root hair formation promoting composition. The amount of biosurfactant contained in the root hair formation promoting composition precursor is preferably 50% by mass or less, and more preferably 20% by mass or less, based on 100% by mass of the root hair formation promoting composition precursor. The amount of agriculturally acceptable carrier (preferably a liquid carrier) contained in the root hair formation promoting composition precursor is preferably 20% by mass or more, and more preferably 40% by mass or more, based on 100% by mass of the root hair formation promoting composition precursor.

<How to grow plants>
As an embodiment of the present disclosure, there is a method for cultivating a plant, in which the composition for promoting root hair formation is applied to a plant or soil. As a method for cultivating a plant, it is preferable to spray the composition for promoting root hair formation on the leaves of the plant.

When spraying on the leaves of plants, the composition for promoting root hair formation is preferably sprayed in an amount such that the amount of biosurfactant in the composition for promoting root hair formation is preferably 5 g to 200 g, more preferably 10 g to 100 g, per 10 a of plant cultivation area.

When the composition for promoting root hair formation is sprayed on the leaves of a plant, there is no particular restriction on the time of spraying, and for example, it can be sprayed once or multiple times at any time after germination and before harvesting the plant.

<Plants>
In the present disclosure, the plant is not particularly limited, but is preferably a crop plant. Examples of crop plants include corn, wheat, barley, rye, oats, rice, soybean, canola (rapeseed), cotton, sunflower, sugar beet, potato, tobacco, broccoli, lettuce, cabbage, spinach, komatsuna, cauliflower, coconut, tomato, cucumber, eggplant, melon, pumpkin, okra, pepper, watermelon, carrot, radish, onion, leek, fruit trees, ornamental plants, grass, and pasture. In addition, one of the preferred aspects is that the plant is a legume. It is known that legume plants increase their yield by forming many root nodules, and an increase in root hairs allows an increase in the number of root nodules attached, which in turn increases the number of root nodules. Therefore, it is preferable that the plant is a legume.

The present embodiment will be described below with reference to examples, but the present disclosure is not limited to these examples.

In the present embodiment, "SF" means Surfactin Na (product name: Kaneka Surfactin, manufactured by Kaneka), "KVP" means soybean meal hydrolysate (a peptide-based material produced by fermenting soybean meal with Bacillus according to APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, pp. 243-247), "SL" means sophorolipid (prepared according to Journal of Oleo Science, 60, (5) pp. 267-273 (2011)), "RL" means rhamnolipid (manufactured by AGAE technology), "SILWET L-77" means a synthetic surfactant manufactured by MOMENTIVE, and "Tween 20" means a synthetic surfactant manufactured by FUJIFILM Wako Pure Chemical Corporation.

[Experimental Example 1]
The following amounts of SF, SL, or RL were dissolved in water to prepare solutions (nodule formation-promoting compositions) for each of the following treatment groups. The solutions were used in an amount of 800 μL/100 g seed to coat soybean (Fukuyutaka) seeds to obtain coated seeds.
Culture soil was filled into 9 cm polypots, and three coated seeds were sown in each pot.

After sowing, the plants were cultivated in a greenhouse set at 28° C. (14 h) during the day and 18° C. (10 h) at night.
Seven days after sowing, the soybeans in each pot were thinned to one plant, and 4.3 ml of a mixture of root nodule bacteria materials (Rhizobium and Azospirillum) in water at a rate of 10 g/L was irrigated into each pot.

After 21 days from the irrigation, the roots were washed with water to remove the soil so as not to remove the nodules, and then the nodules formed on the roots were collected. Among the collected nodules, those that did not pass through a sieve with a mesh size of 1.18 mm were counted to obtain the number of nodules.
After sowing, each pot was fertilized once a week with 1/2 Hoagland's solution. The experiment was carried out on 8 pots (8 plants) for each treatment.

Several experiments were conducted by changing the type of composition for promoting nodule formation. The sowing date, the date of irrigation, the date on which the number of nodules was investigated, the SF, SL or RL amount of the composition for promoting nodule formation, and the results of the investigation of the number of nodules are shown in Tables 1-1 and 1-2. The number of nodules is shown as a relative value with T1 set at 100, and the number in parentheses shows the average number of nodules actually measured (nodules/plant).

Experimental Example 1 suggests that coating seeds with a composition for promoting nodule formation containing at least one type of biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant promotes the formation of nodules.

[Experimental Example 2]
For each of the treatment zones described below, root nodule bacteria materials (Rhizobium and Azospirillum) were mixed with water at a ratio of 10 g/L, and SF, SL or RL was added in the amounts described below to prepare a solution containing the root nodule bacteria materials and SF, SL or RL (composition for promoting nodule formation).
Culture soil was filled into 9 cm polypots, and three soybean (Fukuyutaka) seeds were sown in each pot.

After sowing, the plants were cultivated in a greenhouse set at 28° C. (14 h) during the day and 18° C. (10 h) at night.
Seven days after sowing, the soybeans in each pot were thinned to one plant, and 4.3 ml of a composition for promoting root nodule formation (in treatment area T1, root nodule bacteria materials (Rhizobium and Azospirillum) were mixed in water at a rate of 10 g/L) was irrigated into each pot.

After 21 days from the irrigation, the roots were washed with water to remove the soil so as not to remove the nodules, and then the nodules formed on the roots were collected. Among the collected nodules, those that did not pass through a sieve with a mesh size of 1.18 mm were counted to obtain the number of nodules.
After sowing, each pot was fertilized with 1/2 Hoagland's solution (50 mL) once a week. The experiment was carried out on 8 pots (8 plants) for each treatment.

Several experiments were conducted by changing the type of composition for promoting nodule formation. The sowing date, irrigation date, date when the number of nodules was investigated, composition for promoting nodule formation, and the results of the investigation of the number of nodules are shown in Tables 2-1 and 2-2. The number of nodules is shown as a relative value with T1 set at 100, and the number in parentheses shows the average number of nodules actually measured (nodules/plant).

Experimental Example 2 suggests that irrigation with a composition for promoting nodule formation containing at least one type of biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants promotes the formation of nodules.

[Experimental Example 3]
The following amounts of SF, SL, or RL and/or KVP were dissolved in water to prepare solutions (nodule formation-promoting compositions) for each of the following treatment groups. The solutions were used in an amount of 800 μL/100 g seed to coat soybean (Fukuyutaka) seeds to obtain coated seeds.
Culture soil was filled into 9 cm polypots, and three coated seeds were sown in each pot.

After sowing, the plants were cultivated in a greenhouse set at 28° C. (14 h) during the day and 18° C. (10 h) at night.
Seven days after sowing, the soybeans in each pot were thinned to one plant, and 4.3 mL of a mixture of root nodule bacteria materials (Rhizobium and Azospirillum) in water at a rate of 10 g/L was irrigated into each pot.

After 21 days from the irrigation, the roots were washed with water to remove the soil so as not to remove the nodules, and then the nodules formed on the roots were collected. Among the collected nodules, those that did not pass through a sieve with a mesh size of 1.18 mm were counted to obtain the number of nodules.
After sowing, each pot was fertilized with 1/2 Hoagland's solution (50 mL) once a week. The experiment was carried out on 8 pots (8 plants) for each treatment.

Several experiments were conducted by changing the type of composition for promoting nodule formation. The results of the investigation of the SF, SL or RL and/or KVP amount of the composition for promoting nodule formation, and the number of nodules are shown in Tables 3-1 and 3-2. The number of nodules is shown as a relative value with T1 set at 100, and the number in parentheses indicates the average number of nodules (per plant) actually measured.

Experimental Example 3 suggests that coating seeds with a composition for promoting nodule formation containing at least one biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants and a soybean meal hydrolyzate promotes the formation of nodules.

[Experimental Example 4]
For each of the treatment zones described below, root nodule bacteria materials (Rhizobium and Azospirillum) were mixed with water at a ratio of 10 g/L, and SF, SL or RL was added in the amounts described below to prepare a solution containing the root nodule bacteria materials and SF, SL or RL (composition for promoting nodule formation).
Sterilized, fertilizer-free soil (Heiwa) was filled into 9 cm polypots, and three soybean (Fukuyutaka) seeds were sown in each pot.

After sowing, the plants were cultivated in a greenhouse set at 28° C. (14 h) during the day and 18° C. (10 h) at night.
Seven days after sowing, the soybeans in each pot were thinned to one plant, and 4.3 ml of a composition for promoting root nodule formation (in treatment area T1, root nodule bacteria materials (Rhizobium and Azospirillum) were mixed in water at a rate of 10 g/L) was irrigated into each pot.

After 21 days from the irrigation, the roots were washed with water to remove the soil so as not to remove the nodules, and then the nodules formed on the roots were collected. Among the collected nodules, those that did not pass through a sieve with a mesh size of 1.18 mm were counted to obtain the number of nodules.
After sowing, each pot was fertilized with 1/2 Hoagland's solution (50 mL) once a week. The experiment was carried out on 8 pots (8 plants) for each treatment.

Several experiments were conducted by changing the type of composition for promoting nodule formation. The sowing date, irrigation date, date when the number of nodules was investigated, composition for promoting nodule formation, and the results of the investigation of the number of nodules are shown in Tables 4-1 and 4-2. The number of nodules is a relative value with T1 set to 100, and the number in parentheses is the average number of nodules (nodules/plant) actually measured. Furthermore, fresh nodule weight means the weight of the nodules before drying on the day of collection, and is a relative value with T1 set to 100, and dry underground weight means the weight of roots dried at 80°C for 48 hours, and is a relative value with T1 set to 100.

Experimental Example 4 suggests that the formation of nodules is promoted by irrigating with a composition for promoting nodule formation that contains at least one type of biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants and a soybean meal hydrolyzate.

[Experimental Example 5]
An observation tank was made of 1/2 MS agar medium (0.8% agar) in an acrylic tank. Komatsuna (spring mustard spinach) was sown near the surface of the observation tank.
After sowing, the komatsuna plants were cultivated in an incubator set to a light period (6:00-18:00, temperature 26°C, illuminance 120 μmol/m ² ·s) and a dark period (18:00-6:00, temperature 18°C).
12 to 14 days after sowing, the following liquid (composition for promoting root hair formation) was applied to the leaf surface of the plant body for each treatment group (foliar application) without dripping onto the agar. 4 to 7 days after foliar application, the amount of root hairs at a point 2 cm from the tip of the root was observed under a microscope (microscopic observation). Note that the composition for promoting root hair formation was not applied to the T1 group (control).

As a result of the observations, when compared to the T1 group, those where a significant increase in root hairs was observed were rated as AA, those where an increase in root hairs was observed were rated as BB, those where no increase in root hairs was observed were rated as CC, and those where a total or partial decrease in root hairs was observed were rated as DD.

Several experiments were conducted by changing the sowing date, the date of foliar application, the date of observation, the type of composition for promoting root hair formation, etc. Tables 5 to 9 show the composition for promoting root hair formation, an overview of the procedure, and the results of root hair observation. Figures 1 to 5 show some of the observation results of the experiments shown in Tables 5 to 9 (two plants in each experiment (Figures 1 to 3), two roots of one plant (Figures 1 to 5)).

Experimental Example 5 suggests that a composition for promoting root hair formation, which contains at least one biosurfactant selected from a peptide-type biosurfactant and a sugar-type biosurfactant, and an agriculturally acceptable carrier, is useful for promoting root hair formation. It also suggests that the composition for promoting root hair formation is effective when sprayed on plant leaves. Furthermore, this effect could not be confirmed with synthetic surfactants, suggesting that it is an effect unique to biosurfactants.

[Experimental Example 6]
An observation tank (composition for promoting root hair formation) was prepared in an acrylic tank by adding 1% SF aqueous solution to ½ MS agar medium (agar 0.8%) at the following dilution ratios. Komatsuna (spring Senbatsu) was sown near the surface of the observation tank.

After sowing, the komatsuna plants were cultivated in an incubator set to a light period (6:00-18:00, temperature 26°C, illuminance 120 μmol/m ² ·s) and a dark period (18:00-6:00, temperature 18°C).

Eight days after sowing, the amount of root hairs at a point 1 cm from the root tip was observed under a microscope (microscopic observation). In the T1 group (control), 1/2 MS agar medium without 1% SF aqueous solution was used as the observation tank.

As a result of the observations, when compared to the T1 group, those where a significant increase in root hairs was observed were rated as AA, those where an increase in root hairs was observed were rated as BB, those where no increase in root hairs was observed were rated as CC, and those where a total or partial decrease in root hairs was observed were rated as DD.

Several experiments were conducted by changing the sowing date, the observation date, the type of composition for promoting root hair formation, etc. Table 10 shows the composition for promoting root hair formation, an overview of the procedure, and the results of root hair observation. Figure 6 shows some of the observation results of the experiments shown in Table 10 (two plants for each experiment, and one root from one plant).

Experimental Example 6 suggests that the composition for promoting root hair formation is effective not only when sprayed on the leaves of plants, but also when directly absorbed by the roots.

The upper and/or lower limit values of the numerical ranges described in this specification can be arbitrarily combined to define a preferred range. For example, the upper and lower limit values of the numerical ranges can be arbitrarily combined to define a preferred range, the upper limit values of the numerical ranges can be arbitrarily combined to define a preferred range, and the lower limit values of the numerical ranges can be arbitrarily combined to define a preferred range. In this application, a numerical range expressed using the symbol "~" includes the numerical values written before and after the symbol "~" as the upper and lower limits, respectively.

Throughout this specification, singular expressions should be understood to include the plural concept, unless otherwise specified. Therefore, singular articles (e.g., in the English language, "a," "an," "the," etc.) should be understood to include the plural concept, unless otherwise specified.

Although the present embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and even if there are design changes within the scope that does not deviate from the gist of this disclosure, they are included in this disclosure.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (8)

A composition for promoting nodule formation, comprising at least one biosurfactant selected from peptide-type biosurfactants and sugar-type biosurfactants, and an agriculturally acceptable carrier. The composition for promoting nodule formation according to claim 1, wherein the biosurfactant is contained in an amount of 0.001 to 2% by mass relative to 100% by mass of the composition for promoting nodule formation. The composition for promoting nodule formation according to claim 1, which contains a soybean meal hydrolyzate. The composition for promoting nodule formation according to claim 3, wherein the mass ratio of the biosurfactant to the soybean meal hydrolysate (biosurfactant:soybean meal hydrolysate) is 1:0.05 to 1:150. The composition for promoting nodule formation according to claim 1, wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. The composition for promoting nodule formation according to claim 1, which is used to coat plant seeds with the biosurfactant or to irrigate soil in which plants are grown. A precursor of a composition for promoting nodule formation that becomes the composition for promoting nodule formation according to any one of claims 1 to 6 upon dilution. Seeds treated with a composition for promoting nodule formation according to any one of claims 1 to 6.